Diaphragmatic ultrasonography as a predictor of respiratory muscle fatigue in myasthenia gravis.

INTRODUCTION/AIMS: Easy fatigability, the clinical hallmark of generalized myasthenia gravis (GMG), cannot be detected in a dynamic way. The aim of this study was to assess respiratory function dynamically through diaphragmatic ultrasonography (DUS) in GMG patients. METHODS: GMG patients and controls were recruited in a 1:1 ratio. DUS was performed during one quiet breath and 15 consecutive deep breaths. The diaphragm thicknesses were measured at different positions. Diaphragm thickening fraction (TFdi) and the maximal change in diaphragm thickness (Tmax) during 15 consecutive deep breaths were calculated and transformed to normality, named N-TFdi and N-Tmax, respectively. The percentages of changes in TFdi and Tmax compared with baseline were named ΔTFdi and ΔTmax, respectively. The diagnostic parameter for respiratory muscle fatigue was chosen from ΔTFdi and ΔTmax at different deep breath times according to their ability to distinguish GMG patients from controls and the interrater reliability of TFdi and Tmax. RESULTS: Thirty-four GMG patients and 30 healthy controls were enrolled. N-TFdi and N-Tmax significantly changed as the number of deep breaths increased (p < .001) in GMG patients, but not in controls. ΔTmax of the 15th deep breath (ΔTmax15) was selected as the diagnostic parameter for respiratory muscle fatigue. There were no significant differences in percentage of predicted values of forced vital capacity and arterial partial pressure of carbon dioxide between patients with normal and abnormal ΔTmax15. DISCUSSION: DUS could identify diaphragm fatiguability in GMG patients, which may be more reliable and sensitive in assessment of diaphragm fatigue than conventional methods.

Relative frequencies and clinical features of Guillain-Barré Syndrome before and during the COVID-19 pandemic in North China.

OBJECTIVE: Most studies investigated the relationship between COVID-19 and Guillain-Barré syndrome (GBS) by comparing the incidence of GBS before and during the pandemic of COVID-19. However, the findings were inconsistent, probably owing to varying degrees of the lockdown policy. The quarantine requirements and travel restrictions in China were lifted around December 7, 2022. This study aimed to explore whether the relative frequency of GBS increased during the major outbreak in the absence of COVID-19-mandated social restrictions in China. METHODS: GBS patients admitted to the First Hospital, Shanxi Medical University, from December 7, 2022 to February 20, 2023, and from June, 2017 to August, 2019 were included. The relative frequencies of GBS in hospitalized patients during different periods were compared. The patients with and without SARS-CoV-2 infection within six weeks prior to GBS onset formed the COVID-GBS group and non-COVID-GBS group, respectively. RESULTS: The relative frequency of GBS among hospitalized patients during the major outbreak of COVID-19 (13/14,408) was significantly higher than that before the COVID-19 epidemic (29/160,669, P < 0.001). More COVID-GBS patients (11/13) presented AIDP subtype than non-COVID-GBS cases (10/27, P = 0.003). The mean interval between onset of infective symptoms and GBS was longer in COVID-GBS (21.54 ± 11.56 days) than in non-COVID-GBS (5.76 ± 3.18 days, P < 0.001). CONCLUSIONS: COVID-19 significantly increased the incidence of GBS. Most COVID-GBS patients fell into the category of AIDP, responded well to IVIg, and had a favorable prognosis.

The autism risk gene CNTN4 modulates dendritic spine formation.

Contactin 4 (CNTN4) is a crucial synaptic adhesion protein that belongs to the contactin superfamily. Evidence from both human genetics and mouse models suggests that synapse formation and structural deficits strongly correlate with neurodevelopmental disorders, including autism. In addition, several lines of evidence suggest that CNTN4 is associated with the risk of autism. However, the biological functions of CNTN4 in neural development and disease pathogenesis are poorly understood. In this study, we investigated whether and how CNTN4 is autonomously involved in the development of dendrites and dendritic spines in cortical neurons. Disruption of Cntn4 decreased the number of excitatory synapses, which led to a reduction in neural activity. Truncated proteins lacking the signal peptide, FnIII domains or GPI domain lacked the ability to regulate dendritic spine formation, indicating that CNTN4 regulates dendritic spine density through a mechanism dependent on FnIII domains. Importantly, we revealed that autism-related variants lacked the ability to regulate spine density and neural activity. In conclusion, our study suggests that CNTN4 is essential for promoting dendrite growth and dendritic spine formation and that disruptive variants of CNTN4 interfere with abnormal synapse formation and may increase the risk of autism.

Hyperhomocysteinemia in patients with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.

INTRODUCTION/AIMS: Riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD) is an autosomal recessive disease chiefly caused by variants of ETFDH affecting fatty acid metabolism. In our cohort, hyperhomocysteinemia (HHcy) was common. In this study we aimed to identify the association between RR-MADD and HHcy. METHODS: We performed a retrospective review of 13 patients with RR-MADD. Thirty-three healthy controls were recruited, and logistic regression was used to investigate the association between RR-MADD and HHcy. Muscle tissues from six patients and six controls without myopathies were collected to measure the levels of flavin adenine dinucleotide (FAD), an active form of riboflavin. Whole-exome sequencing was performed to identify the disease-associated variants. RESULTS: The RR-MADD patients had a higher prevalence of HHcy (9 of 12) than controls (6 of 33, P < .001). In the multivariate analysis, RR-MADD was positively related to HHcy (P = .014). Muscular FAD levels were decreased in RR-MADD patients (P = .006). Thirteen variants (8 reported and 5 novel) were identified in ETFDH. Of these, c.250G > A was the most common pathogenic variant with an allelic frequency of 4 of 20. DISCUSSION: HHcy was associated with RR-MADD and may aid in the diagnosis of the disease. Our findings expand the mutational spectrum of RR-MADD.

Diagnostic utility of different dysphagia screening tools to detect dysphagia in individuals with amyotrophic lateral sclerosis.

OBJECTIVE: Dysphagia is a common and serious clinical symptom of amyotrophic lateral sclerosis (ALS). The study aimed to evaluate the diagnostic utility of four dysphagia screening tools in ALS, including the ALS Functional Rating Scale-Revised (ALSFRS-R) bulbar subscale, water-swallowing test (WST), Eating Assessment Tool-10 (EAT-10) and Sydney Swallow Questionnaire (SSQ). METHODS: A total of 68 individuals from First Hospital, Shanxi medical university, were recruited in the study. The ALSFRS-R, WST, EAT-10, SSQ and the gold standard video fluoroscopic swallowing study (VFSS) were performed. The Penetration Aspiration Scale (PAS) during VFSS was assessed to identify unsafe swallowing (PAS ≥ 3) and aspiration (PAS ≥ 6). Receiver operator characteristic curve (ROC) analyses were performed to evaluate the accuracy of the 4 tools. Youden index was used to determine the ideal cut-off value for each tool. RESULTS: Of the patients, 20.59% (14/68) presented unsafety swallowing and 16.18% (11/68) had aspiration. The four tools could effectively identify patients with unsafe swallowing and aspiration. The EAT-10 had the maximum AUC (0.873 and 0.963, respectively) among the tools in the diagnosis of unsafe swallowing and aspiration. To detect unsafe swallowing and aspiration, an EAT-10 score of 6 (sensitivity: 78.6%, specificity: 87.0%) and an EAT-10 score of 8 (sensitivity: 90.9%, specificity: 91.2%), were the most appropriate cut-off points, respectively. CONCLUSIONS: The ALSFRS-R bulbar subscale, WST, EAT-10, and SSQ could effectively identify unsafe swallowing and aspiration in patients with ALS. Of the four tools, the EAT-10 was relatively accurate, safe, and convenient. Further studies including more patients should be conducted to verify the conclusions.

Genetic analysis in Chinese patients with familial or young-onset amyotrophic lateral sclerosis.

OBJECTIVE: The aim of our study was to investigate the genetic characteristics in patients with familial or young-onset amyotrophic lateral sclerosis (ALS) in a Chinese center. METHODS: Patients with familial or young-onset (age of onset < 45 years old) ALS were reviewed. The clinical data was collected. Whole-exome sequencing was performed to identify the disease-associated variants. Single-nucleotide variants and small insertions/deletions were further predicted with silico tools and compared to the Single Nucleotide Polymorphism Database, Exome Aggregation Consortium, and the 1000 Genomes Project. The evolutionary conservations were estimated, and the structures of proteins were constructed by Swiss-Model server. Immunohistochemistry was used to confirm the misfolded SOD1 protein. RESULTS: Three familial ALS and 5 young-onset ALS were enrolled. Genetic analysis identified related variants of SOD1 (4/6, 66.7%), FUS (1/6, 16.7%), and NEK1 (1/6, 16.7%) in 6 patients. Three of them were familial probands (3/3, 100%), and the others were sporadic young-onset patients (3/5, 60%). NEK1 c.290G > A mutation (NM_012224.2 exon4) in a patient with familial ALS and SOD1 c.362A > G mutation (NM_000454 exon5) in a young-onset ALS patient were novel. The novel mutations were predicted to be deleterious, affected evolutionarily highly conserved amino acid residue and the formation of hydrogen bonds between the mutated site and its surrounding amino acid residues. Misfolded SOD1 protein was identified in patient with SOD1 c.362A > G mutation. CONCLUSIONS: Two novel mutations were detected in our patients. Patients with familial or young-onset ALS often carried related gene mutations, and genetic sequencing should be thus routinely performed.

SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis.

High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGF-ß pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGF-ß signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGF-ß signalling and ECM synthesis in the KO cells. The fact that TGF-ß signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGF-ß signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGF-ß signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGF-ß signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.

Biallelic loss-of-function variants in NEMF cause central nervous system impairment and axonal polyneuropathy.

We aimed to detect the causative gene in five unrelated families with recessive inheritance pattern neurological disorders involving the central nervous system, and the potential function of the NEMF gene in the central nervous system. Exome sequencing (ES) was applied to all families and linkage analysis was performed on family 1. A minigene assay was used to validate the splicing effect of the relevant discovered variants. Immunofluorescence (IF) experiment was performed to investigate the role of the causative gene in neuron development. The large consanguineous family confirms the phenotype-causative relationship with homozygous frameshift variant (NM_004713.6:c.2618del) as revealed by ES. Linkage analysis of the family showed a significant single-point LOD of 4.5 locus. Through collaboration in GeneMatcher, four additional unrelated families' likely pathogenic NEMF variants for a spectrum of central neurological disorders, two homozygous splice-site variants (NM_004713.6:c.574+1G>T and NM_004713.6:c.807-2A>C) and a homozygous frameshift variant (NM_004713.6: c.1234_1235insC) were subsequently identified and segregated with all affected individuals. We further revealed that knockdown (KD) of Nemf leads to impairment of axonal outgrowth and synapse development in cultured mouse primary cortical neurons. Our study demonstrates that disease-causing biallelic NEMF variants result in central nervous system impairment and other variable features. NEMF is an important player in mammalian neuron development.

Structure and allosteric coupling of type â ¡ antitoxin CopASO.

Toxin-antitoxin (TA) systems play critical roles in the environment adaptation of bacteria. Allosteric coupling between the N-terminal DNA-binding domain and the C-terminal toxin-binding domain of antitoxins contributes to conditional cooperativity in the functioning of type II TA. Herein, using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC), the structure and DNA binding of CopASO, a newly identified type II antitoxin in Shewanella oneidensis, were investigated. Our data show that CopASO is a typical RHH antitoxin with an ordered N-terminal domain and a disordered C-terminal domain, and furthermore indicate that the C-terminal domain facilitates DNA binding of the N-terminal domain, which in turn induces the C-terminal domain to fold and associate.

The male-to-female ratio in late-onset multiple acyl-CoA dehydrogenase deficiency: a systematic review and meta-analysis.

BACKGROUND: Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common lipid storage myopathy. There are sex differences in fat metabolism and it is not known whether late-onset MADD affects men and women equally. METHODS: In this systematic review and meta-analysis, the PubMed, Embase, Web of Science, CNKI, CBM, and Wanfang databases were searched until 01/08/2023. Studies reporting sex distribution in patients with late-onset MADD were included. Two authors independently screened studies for eligibility, extracted data, and assessed risk of bias. Pre-specified outcomes of interest were the male-to-female ratio (MFR) of patients with late-onset MADD, the differences of clinical characteristics between the sexes, and factors influencing the MFR. RESULTS: Of 3379 identified studies, 34 met inclusion criteria, yielding a total of 609 late-onset MADD patients. The overall pooled percentage of males was 58% (95% CI, 54-63%) with low heterogeneity across studies (I2 = 2.99%; P = 0.42). The mean onset ages, diagnostic delay, serum creatine kinase (CK), and allelic frequencies of 3 hotspot variants in ETFDH gene were similar between male and female patients (P > 0.05). Meta-regressions revealed that ethnic group was associated with the MFR in late-onset MADD, and subgroup meta-analyses demonstrated that East-Asian patients had a higher percentage of male, lower CK, and higher proportion of hotspot variants in ETFDH gene than non-East-Asian patients (P < 0.05). CONCLUSIONS: Male patients with late-onset MADD were more common than female patients. Ethnicity was proved to be a factor influencing the MFR in late-onset MADD. These findings suggest that male sex may be a risk factor for the disease.

Efgartigimod in the treatment of Guillain-Barré syndrome.

BACKGROUND: Guillain-Barré Syndrome (GBS) is caused by immunoglobulin G (IgG) autoantibodies. Efgartigimod, a human IgG antibody Fc fragment that acts as a natural ligand for the FcRn, can increase IgG degradation, which thus may be a promising therapeutic drug for GBS. CASE PRESENTATION: The two patients presented with postinfectious and acute flaccid paralysis. On admission, they were bedridden. Nerve conduction studies indicated peripheral neuropathy. GBS was suspected and they are treated with two doses of efgartigimod (10 mg/kg) within 5 days. Their muscle strength improved gradually and 4 weeks after the initial dose, they could walk independently. Following the first dose, Patient 1 complaint of muscle soreness, which subsided the next morning. Patient 2 was intubated due to respiratory failure the day after the initial dose, and did not report other adverse effects. DISCUSSION: In GBS patients, two doses of efgartigimod (10 mg/kg) were effective in rapidly improving muscle strength, with a satisfactory safety profile. The findings suggest a potential role for efgartigimod in modifying the disease process in GBS patients. CONCLUSION: Efgartigimod seems effective and safe in the treatment of GBS. This study indicates the potential role of efgartigimod as a novel treatment option for GBS. Well-designed clinical trials should be conducted.

A novel autosomal dominant ERLIN2 variant activates endoplasmic reticulum stress in a Chinese HSP family.

OBJECTIVE: Hereditary spastic paraplegia (HSP) has been reported rarely because of a monoallelic variant in ERLIN2. The present study aimed at describing a novel autosomal dominant ERLIN2 pedigree in a Chinese family and exploring the possible mechanism of HSP caused by ERLIN2 variants. METHODS: The proband and his family underwent a comprehensive medical history inquiry and neurological examinations. Whole-exome sequencing was performed on the proband, and Sanger sequencing was performed on some family members. HeLa cell lines and mouse primary cortical neurons were used for immunofluorescence (IF) and reverse transcription-PCR (RT-PCR). RESULTS: Seven patients were clinically diagnosed with pure spastic paraplegia in four consecutive generations with the autosomal dominant inheritance model. All patients presented juvenile-adolescent onset and gradually worsening pure HSP phenotype. Whole-exome sequencing of the proband and Sanger sequencing of all available family members identified a novel heterozygous c.212 T>C (p.V71A) variant in exon 8 of the ERLIN2 gene. The c.212 T>C demonstrated a high pathogenic effect score through functional prediction. RT-PCR and IF analysis of overexpressed V71A revealed an altered ER morphology and increased XBP-1S mRNA levels, suggesting the activation of ER stress. Overexpression of V71A in primary cultured cortical neurons promoted axon growth. INTERPRETATION: The novel c.212 T>C heterozygous variant in human ERLIN2 caused pure HSP. Moreover, c.212 T>C heterozygous variant in ERLIN2 increased ER stress and affected axonal development.

GGC repeat expansions in NOTCH2NLC causing a phenotype of lower motor neuron syndrome.

A genetic diagnosis cannot be made in a considerable proportion of patients with hereditary lower motor neuron (LMN) syndromes. The GGC repeat expansion in the 5'untranslated region (5'UTR) of NOTCH2NLC gene has been reported to be associated with a group of NOTCH2NLC-related repeat expansion disorders (NRED), including amyotrophic lateral sclerosis (ALS). The relationship between the mutation and LMN syndromes has not been reported previously. Here, we identified the GGC repeat expansions of NOTCH2NLC in a Chinese familial patient with LMN syndrome, presenting with slowly progressive weakness of four limbs. Needle electromyography revealed evidence of acute denervation and chronic neurogenic changes. Cognition and brain MRI were normal. Initial whole-exome sequencing by next generation sequencing revealed negative results. However, repeat-primed polymerase chain reaction performed on the proband showed a pathogenic GGC expansion in the 5'UTR of NOTCH2NLC and long-read sequencing subsequently revealed 248 GGC repeats. The mutation was co-segregated with the clinical phenotype in the family. Immunofluorescent studies identified p62-positive protein deposits in the intranuclear inclusions in myofibers. The GGC repeat expansion in NOTCH2NLC is associated with a new phenotype of hereditary LMN syndrome. As a result, NOTCH2NLC genotyping should be performed in patients with hereditary LMN syndromes.

Type II Alexander disease with fragile X mental retardation 1 gene mutation.

Alexander disease (AxD) is a rare, autosomal dominant genetic disorder with an incidence of approximately 1 in 27,00.000. It is caused by a missense mutation in the GFAP gene encoding the glial fibrillary acidic protein. Fragile X-associated tremor/ataxia syndrome (FXTAS) is an X-linked dominant genetic disease, usually caused by a pre-mutation: an unmethylated expansion in the range of 50-200 CGG repeats in the fragile X mental retardation 1 (FMR1) gene. The clinical manifestations of these two diseases are complex and have some similarities. Both type II AxD and FXTAS may have ataxia as the first symptom. Here, we describe a case of type II AxD with ataxia as the first symptom accompanying a hemizygous mutation in the FMR1 gene (NM_001185081, exon13, c 0.1256C>T, p.T419M, g 0.147026507C>T). A sporadic genetic mutation led us to misdiagnose the patient with FXTAS initially. Whole-genome sequencing confirmed a heterozygous mutation in the GFAP gene (NM_002055.5, exon4, c 0.1158C>A, p.N386K, g 0.6310C>A). This report indicates that when the patient's clinical manifestation is ataxia, and imaging results suggest that the midbrain, medulla oblongata, and other subcerebellar structures are atrophied, AxD should be considered. Whole-genome sequencing is thus feasible to avoid missed diagnoses and misdiagnoses.

Rare NRXN1 missense variants identified in autism interfered protein degradation and Drosophila sleeping.

NRXN1 is involved in synaptogenesis and have been implicated in Autism spectrum disorders. However, many rare inherited missense variants of NRXN1 have not been thoroughly evaluated. Here, functional analyses in vitro and in Drosophila of three NRXN1 missense mutations, Y282H, L893V, and I1135V identified in ASD patients in our previous study were performed. Our results showed these three mutations interfered protein degradation compared with NRXN1-WT protein. Expressing human NRXN1 in Drosophila could lead to abnormal circadian rhythm and sleep behavior, and three mutated proteins caused milder phenotypes, indicating the mutations may change the function of NRXN1 slightly. These findings highlight the functional role of rare NRXN1 missense variants identified in autism patients, and provide clues for us to better understand the pathogenesis of abnormal circadian rhythm and sleep behavior of other organisms, including humans.

Novel mutation of EDA causes new asymmetrical X-linked hypohidrotic ectodermal dysplasia phenotypes in a female.

Hypohidrotic ectodermal dysplasia (HED) is a rare hereditary disorder that affects tissues derived from the ectoderm including hair, teeth and sweat glands. EDA is the major causative gene of HED. This study recruited a Chinese family with HED, including a male proband and his mother with a fetus. The proband had typical clinical features of HED and the mother had identical but milder features. Interestingly, some phenotypes of the mother appeared asymmetrically between the right and left side of the body that were not reported in previous studies. Targeted sequencing was performed in the proband and a novel frame-shift mutation (NM_001399.4: c.381_382delinsG, p.Q128Rfs*9) in EDA was found. Sanger sequencing validated the mutation and identified the same mutation in the mother. Our study expands the clinical and genetic spectrum of EDA-related disorders and reports new asymmetrical phenotypes in a female.

Disruptive mutations in TANC2 define a neurodevelopmental syndrome associated with psychiatric disorders.

Postsynaptic density (PSD) proteins have been implicated in the pathophysiology of neurodevelopmental and psychiatric disorders. Here, we present detailed clinical and genetic data for 20 patients with likely gene-disrupting mutations in TANC2-whose protein product interacts with multiple PSD proteins. Pediatric patients with disruptive mutations present with autism, intellectual disability, and delayed language and motor development. In addition to a variable degree of epilepsy and facial dysmorphism, we observe a pattern of more complex psychiatric dysfunction or behavioral problems in adult probands or carrier parents. Although this observation requires replication to establish statistical significance, it also suggests that mutations in this gene are associated with a variety of neuropsychiatric disorders consistent with its postsynaptic function. We find that TANC2 is expressed broadly in the human developing brain, especially in excitatory neurons and glial cells, but shows a more restricted pattern in Drosophila glial cells where its disruption affects behavioral outcomes.

Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission.

RNA binding proteins are key players in posttranscriptional regulation and have been implicated in neurodevelopmental and neuropsychiatric disorders. Here, we report a significant burden of heterozygous, likely gene-disrupting variants in CSDE1 (encoding a highly constrained RNA binding protein) among patients with autism and related neurodevelopmental disabilities. Analysis of 17 patients identifies common phenotypes including autism, intellectual disability, language and motor delay, seizures, macrocephaly, and variable ocular abnormalities. HITS-CLIP revealed that Csde1-binding targets are enriched in autism-associated gene sets, especially FMRP targets, and in neuronal development and synaptic plasticity-related pathways. Csde1 knockdown in primary mouse cortical neurons leads to an overgrowth of the neurites and abnormal dendritic spine morphology/synapse formation and impaired synaptic transmission, whereas mutant and knockdown experiments in Drosophila result in defects in synapse growth and synaptic transmission. Our study defines a new autism-related syndrome and highlights the functional role of CSDE1 in synapse development and synaptic transmission.

Inherited and multiple de novo mutations in autism/developmental delay risk genes suggest a multifactorial model.

Background: We previously performed targeted sequencing of autism risk genes in probands from the Autism Clinical and Genetic Resources in China (ACGC) (phase I). Here, we expand this analysis to a larger cohort of patients (ACGC phase II) to better understand the prevalence, inheritance, and genotype-phenotype correlations of likely gene-disrupting (LGD) mutations for autism candidate genes originally identified in cohorts of European descent. Methods: We sequenced 187 autism candidate genes in an additional 784 probands and 85 genes in 599 probands using single-molecule molecular inversion probes. We tested the inheritance of potentially pathogenic mutations, performed a meta-analysis of phase I and phase II data and combined our results with existing exome sequence data to investigate the phenotypes of carrier parents and patients with multiple hits in different autism risk genes. Results: We validated recurrent, LGD, de novo mutations (DNMs) in 13 genes. We identified a potential novel risk gene (ZNF292), one novel gene with recurrent LGD DNMs (RALGAPB), as well as genes associated with macrocephaly (GIGYF2 and WDFY3). We identified the transmission of private LGD mutations in genes predominantly associated with DNMs and showed that parental carriers tended to share milder autism-related phenotypes. Patients that carried DNMs in two or more candidate genes show more severe phenotypes. Conclusions: We identify new risk genes and transmission of deleterious mutations in genes primarily associated with DNMs. The fact that parental carriers show milder phenotypes and patients with multiple hits are more severe supports a multifactorial model of risk.